Western Blot analysis of Erk signaling pathways and qRT-PCR analysis of gene expression in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. (A) Representative blot and (B) quantification showing phosphorylation of Erk and Erk1/2 protein expression at different time points during EB development. Data are shown as mean ± SEM. N = 3; *P < .05. (C) qRT-PCR analysis of mRNA levels of genes (BMPr2, Smad8, Smad3, Smad7) involved in BMP pathways in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. D0 represents undifferentiated ESCs. Graphs in panel C were plotted in logarithmic scale. Data are relative to SIRT1^+/+ D0 control and mRNA levels of each gene were compared between SIRT1^+/+ and SIRT1^−/− EBs at each time point. Results were of 3 independent experiments performed in triplicate. *P < .05.